The long-term goal of this project is to understand the molecular mechanisms responsible for the regulation of mitochondrial pyruvate dehydrogenase complex (PDC). The reaction catalyzed by PDC plays a central role in general metabolism providing a source of carbon for the major energy-generating and biosynthetic pathways such as the Citric Acid Cycle, lipogenesis, and the biosynthesis of cholesterol. The activity of mammalian PDC is regulated through a reversible phosphorylation/dephosphorylation cycle catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. Growing evidence strongly suggests that control over the phosphorylation state of PDC becomes critically important in starvation, metabolic acidosis, ischemia, and diabetes. The major thrust of this application is to understand the molecular mechanisms responsible for the re-activation of PDC in phosphatase reaction. Previously, we have demonstrated that dephosphorylation of PDC is catalyzed by two closely related phosphatases (PDP1 and PDP2) that are markedly different with respect to their tissue distribution, enzymatic activities and regulation. We have also found that PDP2 protein is down-regulated in diabetes thereby contributing to the unwanted inactivation of PDC, which prevents the aerobic oxidation of carbohydrate fuels. Recently, we obtained the first evidence indicating that, in mitochondria, PDP1 and PDP2 interact with different accessory or targeting subunits. Based on these preliminary data, we hypothesize that the regulation of phosphatase activity in vivo occurs largely through specific protein-protein interactions. In the present application, this major working hypothesis will be investigated through the following specific aims: 1) to isolate and identify the interacting partners of PDP1 and PDP2; 2) to establish the molecular mechanisms of targeting and regulation of PDP1; 3) to establish the molecular mechanisms of targeting and regulation of PDP2; and 4) to elucidate the molecular mechanism of long-term regulation of PDP2 in diabetes. Inactivation of pyruvate dehydrogenase complex due to its hyperphosphorylation has detrimental effect in metabolic acidosis, ischemia, and diabetes. Uncovering the molecular mechanisms responsible for the reactivation of pyruvate dehydrogenase complex carried out by pyruvate dehydrogenase phosphatases 1 and 2 is critical for development of new therapeuticals.